Process for separating proteins fibrinogen, factor XIII and biological glue from a solubilized plasma fraction and for preparing lyophilised concentrates of said proteins

ABSTRACT

Process for separating proteins fibrinogen, Factor XIII and biological glue from a solubilized plasma fraction and for preparing lyophilized concentrates of said proteins.

This application is a Divisional Application of copending applicationSer. No. 11/477,212 filed on Jun. 28, 2006, which claims priority toApplication No. 05 06640 filed in France on Jun. 29, 2005. The entirecontents of all of the above applications are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention is related to a process for separating proteinsfibrinogen, Factor XIII and biological glue, and for preparing highlypurified lyophilised concentrates of said proteins, and is also relatedto said lyophilised concentrates capable to be obtained by the process.

BACKGROUND OF THE INVENTION

Fibrinogen is an essential protein for the blood coagulation because itspolymerisation to insoluble fibrin, which is formed at the end of thereaction cascade governing the coagulation, leads to the formation of aclot, blocking the vascular gap, responsible for the bleeding. Theformation of the clot is essential to stop the bleeding. Further, thefibrin formed on the wound level forms a fibrillary network ensuring thetissue repair (wound healing).

Congenital fibrinogen deficiencies can lead to serious diseases. Inorder to treat these deficiencies, it is necessary that fibrinogenconcentrates, which can be administered to patients under treatment, areavailable. Other pathologies can also be treated by administering offibrinogen, especially in cases of massive blood losses (surgery,traumas, etc.), or following to a disseminated intravascular coagulationsyndrome (CIVD).

Moreover, biological glues, activated by thrombin, containing fibrinogenas the major component, and Factor XIII (FXIII), are efficiently used intissue repair in clinics, such as skin transplantation, nervous andarterial sutures, as described, for example, in patents EP 0 305 243, FR2 448 900 and FR 2 448 901. The presence of Factor XIII ortransglutaminase in these products contributes to the stabilization offibrin by formation of intercatenary covalent bindings which make itinsoluble. In some cases, these products are obtained by means of rathercomplex fibrinogen production processes, which require an externalsupply of purified Factor XIII, in order to be able to perform theirtherapeutic function.

Therefore, production of fibrinogen, biological glues and Factor XIIIconcentrates, especially for therapeutic uses, requires purificationtechniques leading to these products, which are not only sufficientlyfree of various contaminants, such as the accompanying or coprecipitatedproteins, antibodies or proteases but, in addition, their viral safetyis increased.

The isolation of fractions enriched in fibrinogen, possibly containingFXIII, from plasma, is well known and first described by Cohn andNitschmann (Cohn et al, J. Am. Chem. Soc., 68, 459, 1946 and Kistler etal, Vox Sang., 7, 1962, 414-424). More recent methods combineprecipitating techniques of different plasma sources with filtration,chromatography, viral inactivation techniques, etc. The followingpatents and patent applications can be cited as examples: EP 0 359 593,U.S. Pat. No. 5,099,003, EP 0 305 243, FR 2 448 900 and FR 2 448 901.

Nevertheless, different processes yielding concentrates or compositionseither of fibrinogen, as described in the patent application EP 1 457497, or of biological glue, for example according to the patent EP 0 771324, or enriched in fibrinogen containing further associated proteinssuch as FXIII, Factor VIII, fibronectin, Factor von Willebrand etc.(especially U.S. Pat. No. 6,121,232) are carried out.

These processes, however, involve the use of separate production, linesconsequently using different methods employing several sources of rawmaterials for obtaining these considered proteins. Furthermore,depending upon the case, these methods can involve expensivechromatographic substrates, such as affinity gels based on chelatedmetals (WO 2004/007533) liable to release residual metals into theeluate, which can lead to unwanted reactions with the proteins (forexample oxidation). This creates problems of clumsiness of the carryingout on industrial scale, when these three purified active principles areneeded together. These problems are even more obvious when the differentthus obtained proteins are to be subjected to a viral inactivationand/or viral and other unwanted contaminants, such as prions, removaltreatment.

To this end, some classical viral inactivation treatments implementing aheat treatment, such as pasteurisation at 60° C., for 20 hours, in thepresence of protecting stabilizers, and a chemical treatment, such assolvent-detergent, intended to make the above concentrates compatiblewith therapeutic use, do not allow to inactivate completely the viruses,especially non-enveloped viruses (parvovirus B19, hepatitis A and B,etc.).

In order to find a solution to this drawback, use is currently made ofmore efficient viral inactivation processes, such as dry heat treatmentunder harsh conditions (80° C., 72 h). This step requires theincorporation of a suitable stabilizing formulation offering conditionssuch as, for example, the fibrinogen stabilization in this step, whilethe viruses are being destroyed. Such a formulation is thesubject-matter of a patent application FR 04 02001 filed by theApplicant. However, this formulation can be applied to the stabilizationof a defined protein and not of the accompanying proteins,characteristics of which are different of those of fibrinogen.

The filtration techniques, especially the nanofiltration using filterswith a porosity of 35 nm, and even less, have also been carried out inorder to remove viruses. However, this technique cannot be efficientlyused without controlling the physical and chemical parametersinfluencing the recovery output of compounds to be filtered, and this byavoiding the clogging of the filter and the passage of various virusesand contaminants. These parameters, such as ionic strength, pH of thesolution, and filtration process conditions, as well, lay down thespecific process conditions which depend also on the nature of thecompound(s) contained in the solution to be filtered. Although thepatent applications EP 1 348 445 A1, EP 1 161 958 A1 and WO 99/23111disclose the nearly total removal, of very small sized non-envelopedviruses present in the protein solutions, such as hepatitis A, bynanofiltration, making use of filters of 15 nm, however, the risk oftransmission of unwanted viruses or prions is always present.

In order to avoid this risk, a double or even a triple viralinactivation and/or removal combining at least two of the abovementioned techniques can be performed, as described for example in thepatent application WO 2004/007533. When such treatments are combined,then it is essential to choose, depending upon the viral inactivationmethod, the virucidal excipients and/or protecting stabilizers which arenot exerting a concomitant deleterious effect, as for example on theabove mentioned physical and chemical parameters governing thenanofiltration.

SUMMARY OF THE INVENTION

Therefore, the Applicant, investigated the development of a process forseparating fibrinogen, Factor XIII and biological glue activated bythrombin meeting a double objective. On one hand, the development of aunique process which allows to obtain together concentrates of thesefreeze-dried and highly purified proteins from a single plasmatic rawmaterial containing fibrinogen and Factor XIII, and, on the other hand,this process should be compatible with at least one viral inactivationand/or viral and other unwanted contaminants (polymers, aggregates,prion) removal treatment, as well.

Therefore, the present invention is related to a process for separatingproteins fibrinogen, Factor XIII and biological glue from a solubilizedplasma fraction, based on fibrinogen and Factor XIII, and for preparingfreeze-dried concentrates of said proteins comprising the followingsteps of:

-   a) chromatographic purification comprising the steps of:

loading an anion exchanger of weak base type with the said solubilizedfraction, said exchanger being previously equilibrated with a bufferhaving a predetermined ionic strength of an alkaline pH, thus allowingthe retention of the biological glue,

elution of the biological glue by increasing of the ionic strength ofthe said buffer,

-   a) separation of FXIII from fibrinogen by addition to at least a    part of the biological glue eluate of at least one chemical agent    precipitating the FXIII, and recovery of the resulting supernatant    solution of purified fibrinogen, and-   b) diafiltration of the fibrinogen, biological glue and    resolubilized FXIII solutions, followed by a freeze-drying of said    solutions.

Thus, the Applicant found that freeze-dried, highly purified,fibrinogen, Factor XIII and biological glue concentrates, free ofcoprecipitated proteins and of unwanted contaminants, can be obtained onindustrial scale by means of a unique flexible process which allows,depending upon the needs, to adjust optimally the production of eachconsidered compound, while ensuring the optimal profitability of the rawmaterial. Such a simple, rapid and low cost process is easy to carry outon industrial scale, and yields an increased optimisation of variousproduction flow-charts.

Moreover, depending upon the used raw material and the intendedapplication, additional process steps oil viral inactivation and/orremoval are yielding the three concentrates of interest, suitable fortherapeutic use.

The invention is related to a process for separating proteinsfibrinogen, Factor XIII and biological glue from a solubilized plasmafraction and for preparing freeze-dried concentrates of said proteinscomprising the steps of:

-   -   chromatographic purification comprising the steps of loading an        anion exchanger of weak base type with the said solubilized        fraction, previously equilibrated with a buffer of a        predetermined ionic strength of an alkaline pH, which allows to        retain the biological glue, elution of the biological glue by        increasing the ionic strength of the said buffer, and    -   separation of FXIII from fibrinogen by addition to at least one        part of the biological glue eluate of at least one chemical        agent precipitating the FXIII, and recovery of the resulting        purified fibrinogen containing supernatant solution, and    -   diafiltration of the fibrinogen, biological glue and        resolubilized FXIII solutions, followed by a freeze-drying of        said solutions. It is also related to freeze-dried concentrates        of the said proteins capable to be obtained by carrying out the        process.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the invention, use can be made or several sources of rawmaterials containing fibrinogen and Factor XIII. Thus, these Plasmafractions are obtained by plasma fractionation collected underconditions which are unfavourable for maintaining a sufficient ratio ofFactor VIII, which is a labile protein, following to the Cohn processusing cold alcohol. It is also possible to apply the above mentionedfractionation to a previously solubilized cryoprecipitate, as far as theextraction of Factor VIII is not aimed, for example in the case of acryopreciptate with an expired shelf-life or not meeting the conformityrequirements of the minimal content of Factor VIII, or tocryoprecipitate depleted plasma. So these different sources offibrinogen constitute a precipitate of Cohn fraction I which, afterwashing, is dissolved in any suitable buffer with a neutral pH, known bythose skilled in the art. For example, such a buffer is based on sodiumchloride, trisodium citrate and L-arginine, with a pH of about 7.4,containing each component in a concentration preferably of 0.12 M, 0.01M and 0.05 M, respectively.

Thus the flexibility of the process of the invention is also related tothe variety of raw materials liable to contain extractible fibrinogen,the purification of which yields the three considered concentratesintended for targeted therapeutic uses.

The process can also include, prior to step a) an initialprepurification step of the solubilized plasma fraction by a classicalpretreatment with aluminium hydroxide and/or by a precipitation at lowtemperature. The addition of aluminium hydroxide ensures the removal ofunwanted proteins, such as the Factors II (FII), VII, IX and X (FX).This prepurified fraction can be frozen until being used in order tocarry out the following process steps of the invention.

The chromatographic purification of the solubilized plasma fraction iscarried out on any matrix based on a natural or synthetic polymer, resinor gel, on which are grafted groups of anion exchangers of weak basetype, such as the DEAF. Classical chromatographic substrates of thistype are available under trade names DEAE-Sepharose® CL-69,DEAE-Trisacryl LS, Fractogel TSK-DEAE 650 M or S, DEAE-Macroprep(Bio-Rad, France), etc.

The equilibrating buffer of the anion exchanger presents a predeterminedionic strength and its pH value has to be in the alkaline range.

The equilibrating buffer has an ionic strength typically of less than0.2 and, preferably, is in the range of values of from 0.06 to 0.2. Moreparticularly, the ionic strength is in the range of values of from 0.08to 0.15. It is preferably adjusted by addition of inorganic salts ofalkali or alkali-earth metals or mixtures thereof, most preferably ofinorganic salts of alkali metals, particularly of sodium chloride.

The maximal pH value of the equilibrating buffer is such as to avoid anydenaturation of the considered products, that is of about 10.Advantageously, the pH is in the range of values higher than 7 up to 9,preferably from 7.5 to 8.2. By way of example, this buffer contains aconcentration of 0.06 M of sodium chloride with a pH of 7.9-8.1, andvery preferably can further comprise trisodium citrate in a preferredconcentration of 0.011 M. Any other butter based on sodium chloride oron inorganic salts of alkaline or earth-alkaline metals, comprisingfurther biologically active compounds, which are compatible and notdenaturating the products of interest, can also be used.

When the above solution has been applied to the anion exchanger, thebiological, glue is retained on the substrate. The process can include,prior to the biological glue elution step, a washing step with the saidequilibrating buffer of the anion exchanger until not retained proteinsand contaminants are removed. This washing step allows, by percolationof this buffer on the substrate, the passage into the filtrate of thenot-retained or weakly retained, on the exchanger, proteins present inthe solution containing the fibrinogen, such as immunoglobulins G (IgG),A (IgA) and M (IgM) and albumin, and contaminants, such as the chemicalviral inactivation agents. The washing period of time is determined bymeasuring the optical density (OD) of the filtrate at a wavelength of280 nm. Indeed, a value of OD corresponding to that of the baseline is agood indication that the above mentioned compounds were effectivelyeliminated.

After return to the baseline, the elution of the biological glue iscarried out by increase of the ionic strength of the equilibrating orwashing buffer, the pH of which is preferably set to a value of 7.4-7.6.The value of this ionic strength is selected with the aim of obtainingan efficient elution of the biological glue, making sure that this valuedoes not alter the properties of the considered product. Advantageously,the value of the ionic strength is in the range between 0.5 and 1.3,particularly between 0.9 and 1.1. This increase of the ionic strength iscarried out by addition of any here above defined salt or mixture ofsalts, especially of sodium chloride. Moreover, the elution buffer cancontain further excipients, such as a mixture of components calledmixture A, comprising trisodium citrate (10 to 12 g/l), lysine (1 to 5g/l), glycine (1 to 5 g/l), Tris salt (2 to 5 g/l), arginine (25 à 50g/l) and isoleucine (5 à 15 g/l). The protein concentration in theeluate is of about 4 g/l.

At least one part of the recovered amount of the biological glue eluateis subjected to a treatment in order to separate the FXIII accompanyingthe fibrinogen. This separation is carried out by precipitating of theFXIII by addition, to the eluate to be treated, of a chemicalprecipitating agent, which can be present in form of an aqueous solutionin a concentration allowing to attain the desired effect. Preference isgiven to aqueous solutions based on citrate salts 1 M, as for examplesodium citrate and, especially, trisodium citrate. Thus, a precipitateof FXIII and a supernatant highly enriched in fibrinogen are separated.A very efficient recovery of the FXIII precipitate can be achieved byfiltration on filters of 5 μm.

The so recovered FXIII precipitate is resolubilized, preferably in wateror in a buffer. In particular, it is dissolved in a buffer of mixture A,the pH of which is in the range of 6.9-7.1, so that its concentrationcorresponds to an activity of about 100 times higher than that of normalplasma. As such, the FXIII precipitate can be for example solubilized ina way that its concentration be of about 1 to 5 g of total protein/l.

According to the invention, solutions of biological glue (biologicalglue eluate) and of fibrinogen (supernatant enriched in fibrinogen) canbe concentrated by ultrafiltration, up to contents typically between 15and 25 g of total protein/l determined by classical measuring methodswell known to those skilled in the art.

The three obtained fibrinogen, Factor XIII and biological gluesolutions, optionally concentrated, are subjected to a diafiltrationstep. This step is intended at first to remove the possible excess, onone hand, of the inorganic salt used for obtaining solutions with anionic strength as high as 0.2 M, and, on the other hand, of theprecipitating agent present in the resolubilized precipitate. It shouldbe noted that the presence of important amounts of inorganic salt,necessary for eluting the biological glue, may have a deleterious effecton the efficiency of the freeze-drying process and also on the viralinactivation by dry heating, and on the virus retention ability of asuitable nanofilter. This step can also be necessary in order toincorporate, if need be, suitable excipients, stabilizers and protectingagents, which should allow, on one hand, the dry heating of fibrinogen,of FXIII and of biological glue, avoiding the risk of denaturation, and,on the other hand, the rapid solubilization of the freeze-driedproducts, typically in a period of time of 3 to 8 min. The preferreddiafiltration buffer contains the mixture A, and has a pH in the rangeof 6.9-7.1, in reference to the patent application FR 04 02001 filed bythe Applicant.

It should be noted that an other advantage of the invention is that,according to other preferred embodiments the process, the diafiltrationbuffer comprising the mixture A can already be present as a component ofthe biological glue elution buffer. Thus the carrying out of thediafiltration happens to be simplified and optimised.

Diafiltration buffers of different composition can also be used,depending upon the needs, provided that they meet the above mentionedrequirements.

The here above mentioned ultrafiltration step can also be carried outunder the same conditions at this stage of the process.

The respective solutions, optionally diafiltrated, optionallyconcentrated, are freeze-dried by means of classical methods and undercurrent conditions, that is at a temperature in the range between −40°C. and −30° C., for about 48 hours.

Moreover, the process can include at least one step of viralinactivation and/or viral and the abovementioned contaminants, such asprions, removal treatment. This treatment can be selected from the groupconsisting of the chemical viral inactivation treatment, thenanofiltration and the dry heat viral inactivation treatment.

Thus, this step can be carried out by a classical chemical viralinactivation treatment, preferably consisting of a solvent-detergenttreatment, according to the method described in the patent EN 0 131 740.Preferably, the viral inactivating chemical agents are a mixture ofTween®-TnBP, more preferably, the mixture of Triton® (octoxinol)-TnBP,typical concentrations of which are of 0.3% (v/v) and 1% (p/v)respectively. This viral inactivation can be integrated at any stage ofthe process, but it can be carried out judiciously prior to the step a)of the chromatographic purification step. So it will contribute to anefficient removal of the inactivating agents.

In a preferred embodiment of the process, a nanofiltration step can alsobe provided for, in order to remove the viruses, especiallynon-enveloped viruses and other exogenous contaminants, completing theprevious chemical viral inactivation treatment. Filters of 35 nm can beefficiently used, although other nanometric filters can be used as faras the filtration periods of time and the efficiency of viral retentionare optimised. The nanofiltration is carried out with the eluateobtained in step a) or, if the case arises, with the diafiltratedfibrinogen, biological glue and resolubilized FXIII solutions, prior tofreeze-drying. The judicious choice of chemical parameters of thechromatographic purification, and those of the diafiltration allows aflexibility of the carrying out of nanofiltration without modifying itsperformances.

Finally, the dry heat viral inactivation treatment can be carried out onthe freeze-dried products fibrinogen, biological glue and FXIII underclassical conditions, at 80° C., for 72 hours, in order to inactivatethe non-enveloped viruses, which would not have been inactivated and/orremoved in at least one of the previous viral inactivation, and/orremoval steps.

Further, the dry heated freeze-dried products can be reconstituted in anaqueous medium suitable for clinical use, preferably in purified waterfor injection (PPI), and used directly for intravenous injection.

Moreover, the process of the invention can comprise at least one step ofclarifying filtration in order to remove insoluble particles, and atleast one sterilizing step, these being carried out in a current wayusing filters, for example, of 0.8 to 0.1 μm. Particularly, they arerelated to the solubilized prepurified plasma fraction, the eluate ofbiological glue obtained in the step b) and/or the diafiltratedsolutions of the three compounds of interest.

Thus, the carrying out of the process yields freeze-dried, highlypurified biological glue and fibrinogen concentrates which have arespective content of fibrinogen, compared to the content of totalproteins, of about 90%. Further, the Factor XIII activities in thebiological glue and fibrinogen concentrates are respectively of about 5U/ml and of about 1.5 U/ml.

The obtained Factor XIII concentrate is free of contaminating proteins,and presents an activity, if need be, in the range of values of about 30U/ml to about 700 U/ml, preferably of 100 U/ml to 400 U/ml, dependingupon the concentration obtained by resolubilization of the FXIIIprecipitate and/or after ultrafiltration.

The invention is also related to freeze-dried fibrinogen, biologicalglue and Factor XIII concentrates, obtainable by performing the aboveprocess, characterized in that they comprise the mixture ofdiafiltration buffer components (mixture A). Furthermore, the saidconcentrates can be of therapeutic quality thanks to the integration ofat least one step of viral inactivation and/or viral and contaminantsremoval treatment, selected from the group consisting of the chemicalviral inactivation treatment, the nanofiltration and the dry heat viralinactivation treatment, into the process of the invention.

The following Example illustrates one embodiment of the presentinvention without limiting its scope.

EXAMPLE

1200 l of human cryoprecipitate-depleted plasma are used. This plasma issubjected to a precipitation with ethanol according to Cohn's method,under the conditions known by those skilled in the art, in such a waythat the considered ethanol concentration in the plasma is of 8% (v/v)and the temperature of the obtained mixture is of −3° C.

Further, the thus obtained supernatant and precipitate are subjected toa centrifugation. 10 kg of precipitate are obtained, which is the impureCohn fraction I.

The impure Cohn fraction I (10 kg) is suspended and washed with 300 l ofbuffer <<Blombach>>, comprising a mixture of glycine 1 M, of trisodiumcitrate 0.055 M and of ethanol 6.5% (v/v), at a pH of 6.8.

After centrifugation, 8 kg of purified precipitate paste (purified Cohnfraction I) are recovered then dissolved at a temperature of 37° C., in60 l of buffer constituted of a mixture of sodium chloride 0.12 M, oftrisodium citrate 0.010 M and of arginine 0.05 M, pH 7.4.

The thus obtained precipitate solution is then subjected to apre-purification treatment with alumina gel, at a ratio of 108 g per 1kg of precipitate paste, at a temperature of 25° C., and a pH of6.9-7.1.

As soon as the prepurification treatment is achieved, the solution issubjected to lenticular clarifying filtrations by means of cellulosicfibres filters (Seitz, type E700) of 0.65 μm and to sterile filtrationswith filters of 0.2 μm.

This prepurified solution is subjected to a first viral inactivationtreatment by solvent-detergent in presence of Tween®-TnBP, ofconcentrations respectively of 0.3% (v/v) and 1% (p/v), according to themethod described in EP 0 131 740.

The thus treated prepurified solution is diluted to 50% by addition ofthe required volume of an aqueous trisodium citrate 0.010 M solution.

The thus obtained prepurified solution contains furthermore, besides thefibrinogen and the accompanying Factor XIII, unwanted proteins, such asimmunoglobuins G and albumin, and contaminants, such as Tween®-TnBP. Theefficient removal, thereof is carried out by means of a chromatographicstep.

To this end, the prepurified solution is injected onto a chromatographiccolumn filled with an anion exchanger gel DEAE Macroprep (Bio-Rad,France) previously equilibrated with a buffer constituted of sodiumchloride 0.06 M and trisodium citrate 0.011 M, adjusted to a pH 8.0 M,with an osmolarity of 130-150 mosmolkg⁻¹. Under these conditions,fibrinogen and Factor XIII, constituting the biological glue, areretained on the substrate. The weakly retained or not retained proteinson the substrate are removed into the filtrate, and Tween® and TnBP aswell, by several subsequent washings with the same buffer.

When the DO, measured at 280 nm, falls back to the baseline, the elutionof fibrinogen and Factor XIII, constituting the biological glue, iscarried out by means of an elution buffer containing sodium chloride 1Mand a mixture A′ constituted of trisodium citrate (11.2 g/l), lysine(2.0 g/l), glycine (2.0 g/l), Tris salt (2.40 g/l), arginine (40 g/l)and isoleucine (10 g/l), the pH adjusted to 7.5, with an osmolarity>2000mosmolkg⁻¹.

The thus recovered purified biological glue eluate is treated bynanofiltration on PLANOVA filters (Asahi, Japan) of 35 nm, with asurface of 1 m², in order to remove the viruses, which would have notbeen inactivated by the previous solvent-detergent treatment. The totalprotein content, at this stage of the process, is of about 4.0 g/l ofsolution.

50% of volume of the biological glue eluate are isolated and a solutionof trisodium citrate 1M is added to the remaining volume of eluate inorder to precipitate the Factor XIII. After making sure that the entireamount of Factor XIII was precipitated, isolation and recovery byfiltration on Sartopure filters (Sartorius—France) of 5 μm were carriedout.

The thus recovered FXIII precipitate is solubilized in purified waterfor injection, with a ratio of about 1 g/l.

The biological glue (eluate) and fibrinogen solutions are concentratedby ultrafiltration on Biomax Millipore filters 100 kDa with a surface of5 m² in a way that the protein content of each solution attains 15 g/l.

The concentrated here above solutions and the solution of FXIII aresubjected to a diafiltration on the same filters then those used for thediafiltration against the here above defined mixture A′, with a pH6.9-7.1, an osmolarity of 590-610 mosmolkg⁻¹, allowing to remove thesodium chloride, as well.

After carrying out a sterile filtration on 0.45-0.2 μm filters, 100 mlof each diafiltrated solution were taken and were put into glass vialsin order to carry out a freeze-drying at a temperature between −40° C.and −30° C. for about 48 hours.

The obtained freeze-dried fibrinogen, biological glue and Factor XIIIare subjected to an ultimate step of viral inactivation by dry heatingat 80° C. for 72 hours, and stocked before being used in therapy.

The results of quality controls performed on 3 consecutive batches areshown in the following Table.

Biological Proteins FXIII^(a) glue Fibrinogen Yield (%) — 87 ± 3  93 ±4  FXIII: Ag 31.1 ± 2.1  9.3 ± 0.4 2.6 ± 0.9 (U/ml) Activity of 33.3 ±1.5  5.2 ± 0.4 1.3 ± 0.6 FXIII (U/ml) Fibronectin 0.16 ± 0.03 1.10 ±0.04 1.10 ± 0.02 (mg/ml) IgM (μg/ml)  35 ± 0.4 209 ± 46  183 ± 31  IgG(μg/ml) 6 ± 2 29 ± 6  29 ± 5  IgA (μg/ml) <12 ± 3    55 ± 2  40 ± 4 FII: Ag   3 ± 0.2 1.9 ± 0.6 1.2 ± 0.2 (μg/l) (mU/ml) (mU/ml) Activity of— <1.3 ±  <1.3 ± 0.03   FX (mU/ml) 0.04 Plasminogen — 73 ± 4  73 ± 7 (μg/ml) ^(a)total protein concentration of 1.2 g/l

The invention claimed is:
 1. A process for preparing a freeze-driedfibrinogen concentrate comprising the steps of: a) loading an anionexchanger of weak base type with a Cohn fraction I, said exchanger beingpreviously equilibrated with a first buffer having a predetermined ionicstrength of an alkaline pH, thus allowing the retention of thebiological glue containing fibrinogen and Factor XIII (FXIII), b)eluting the biological glue with a second buffer having an alkaline pHand an ionic strength greater than said first buffer, c) separatingFXIII from fibrinogen by precipitation of FXIII by addition of at leastone chemical agent to the eluted biological glue, wherein the chemicalagent precipitating the FXIII is present in the form of an aqueoussolution based on citrate salts 1 M, d) recovering the supernatantsolution containing fibrinogen, e) concentrating the fibrinogensolution, f) diafiltering the concentrated fibrinogen solution, followedby freeze-drying the fibrinogen solution.
 2. The process according toclaim 1, wherein said first buffer has an ionic strength of less than0.2.
 3. The process according to claim 1, wherein the pH of said firstbuffer is in the range of values higher than 7 up to
 9. 4. The processaccording to claim 1, wherein the process includes, prior to thebiological glue elution step, a washing step with said first buffer ofthe anion exchanger until not retained proteins and contaminants areremoved.
 5. The process according to claim 1, wherein the biologicalglue elution is carried out with said second buffer having an ionicstrength in a range between 0.5 and 1.3, the pH of which is set to avalue of 7.4-7.6.
 6. The process according to claim 5, wherein saidsecond buffer further contains a mixture from 10 to 12 g/l of trisodiumcitrate, from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2to 5 g/l of Tris, from 25 to 50 g/l of arginine and from 5 to 15 g/l ofisoleucine.
 7. The process according to claim 1, wherein thediafiltration is carried out against a buffer containing trisodiumcitrate, lysine, glycine, arginine and isoleucine.
 8. The processaccording to claim 1, further comprising at least one step of viralinactivation and/or viral and contaminants removal treatment, selectedfrom the group consisting of a chemical viral inactivation treatment, ananofiltration and a dry heat viral inactivation treatment.
 9. Theprocess according to claim 8, wherein the nanofiltration is carried outwith the diafiltered fibrinogen, prior to freeze-drying.
 10. The processaccording to claim 1, wherein a concentration step by ultrafiltration iscarried out prior to the step of diafiltration or following to the saidstep, prior to freeze-drying.
 11. The process according to claim 1,including prior to step a), an initial prepurification step of the Cohnfraction I by a classical pretreatment with aluminium hydroxide and/orby a precipitation at low temperature.
 12. A freeze-dried fibrinogenconcentrate, obtained by the process according to claim 1, wherein saidconcentrate contains a mixture from 10 to 12 g/l of trisodium citrate,from 1 to 5 g/l of lysine, from 1 to 5 g/l of glycine, from 2 to 5 g/lof Tris, from 25 to 50 g/l of arginine and from 5 to 15 g/l ofisoleucine.